A construction method and application of UPLC fingerprint spectrum of cistanche or cistanche tubulosa medicinal materials, decoction pieces, standard decoction and formula granules

Abstract

The application provides a construction method of UPLC fingerprint of Cistanche or Cistanche tubulosa medicinal material, decoction piece, standard decoction and formula granules, comprising the following steps: preparing a test sample; preparing the test sample solution; determining the test sample solution by using ultra performance liquid chromatography to obtain the UPLC fingerprint corresponding to the test sample. The construction method of UPLC fingerprint of Cistanche or Cistanche tubulosa medicinal material, decoction piece, standard decoction and formula granules provided by the application realizes good separation of common characteristic peaks, simple elution procedure, smooth baseline of the obtained fingerprint, less noise interference, good characteristic peak shape and high separation degree, provides a basis for quality detection and control of Cistanche and Cistanche tubulosa medicines and preparations, and realizes overall component characterization of Cistanche and Cistanche tubulosa medicines and preparations.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method and application of UPLC fingerprint spectrum of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight medicinal material, decoction piece, standard decoction and formula granule. BACKGROUND

[0002] Cistanche deserticola Y.C.Ma and Cistanche tubulosa (Schenk) Wight are medicinal plants of the genus Cistanche of the family Orobanchaceae, have extremely high medicinal value, can be used as the original plant of Cistanche, and have been included in Chinese Pharmacopoeia.

[0003] Cistanche deserticola Y.C.Ma is a slightly curved, flat cylindrical shape, 3-15 cm long, and 2-8 cm in diameter. The surface is brown or gray-brown, densely covered with fleshy scale leaves arranged in a tile-like manner, and the scale leaf tips are usually broken. The body is hard and slightly flexible, not easy to break, the cross section is brown, with light brown dot-shaped vascular bundles arranged in a wavy ring. The smell is weak, and the taste is sweet and slightly bitter.

[0004] Cistanche tubulosa (Schenk) Wight is a slightly curved, flat spindle or flat cylindrical shape, 5-25 cm long, and 2.5-9 cm in diameter. The surface is brown to black brown. The cross section is granular, gray-brown to gray-brown, with scattered dot-shaped vascular bundles.

[0005] In order to ensure the uniformity and stability of Cistanche deserticola Y.C.Ma and Cistanche tubulosa (Schenk) Wight medicinal material and decoction piece quality, it is necessary to establish a new fingerprint method to control the quality. In the prior art, thin layer chromatography and the like are mainly used to detect Cistanche. However, since Cistanche and Cistanche tubulosa are different species of plants in the same family and genus, they have great similarity, and the detection method of the prior art is difficult to distinguish between the two. SUMMARY

[0006] In view of the above problems in the prior art, the application provides a construction method and application of UPLC fingerprint spectrum of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight medicinal material or decoction piece, which can provide a more accurate analysis method and identification basis for identification of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight medicinal material, decoction piece, standard decoction and formula granule.

[0007] To achieve the above object, the application adopts the following technical solution:

[0008] A construction method of UPLC fingerprint spectrum of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight medicinal material, decoction piece, standard decoction and formula granule, comprising the following steps:

[0009] Prepare a test sample: the test sample is Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight medicinal material, decoction piece, standard decoction or formula granule;

[0010] The test product is prepared into a test product solution;

[0011] The test product solution is determined by using ultra-high performance liquid chromatography to obtain the UPLC fingerprint corresponding to the test product;

[0012] The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane-bonded silica gel is used as the filler, the mobile phase A is acetonitrile, the mobile phase B is 0.1wt% acetic acid solution, and gradient elution is used.

[0013] Preferably, the Cistanche or Cistanche tubulosa decoction piece is a processed product of Cistanche or Cistanche tubulosa medicinal material; the Cistanche or Cistanche tubulosa standard decoction is a freeze-dried powder prepared by processing, decocting, separating, concentrating, and freeze-drying the Cistanche or Cistanche tubulosa medicinal material; and the Cistanche or Cistanche tubulosa formula granule is a granule prepared by processing, extracting, separating, concentrating, drying, granulating, and packaging the Cistanche or Cistanche tubulosa medicinal material.

[0014] Preferably, the solvent of the test product solution is methanol-water. More preferably, it is 50%vol methanol-water.

[0015] More preferably, when the test product is Cistanche or Cistanche tubulosa medicinal material or decoction piece, the method for preparing the test product solution is as follows:

[0016] The test product is heated and refluxed in water for 45 minutes, and after cooling, it is filtered and the filtrate is evaporated to dryness. Then, methanol-water is added, and the mixture is ultrasonically treated for 30-60 minutes. After cooling, it is filtered, and the filtrate is obtained.

[0017] More preferably, when the test product is Cistanche or Cistanche tubulosa standard decoction or formula granule, the method for preparing the test product solution is as follows:

[0018] The test product is placed in methanol-water, and ultrasonically treated for 15-60 minutes. After cooling, methanol-water is added to make up for the weight loss, and the mixture is mixed and then left to stand. The supernatant is filtered, and the filtrate is obtained.

[0019] Preferably, in the test product solution, the ratio of the amount of test product to solvent is 1g:50mL.

[0020] Preferably, the gradient elution program is as follows:

[0021] 0-5 minutes, mobile phase A: 10%vol-13%vol, mobile phase B: 90%vol-87%vol;

[0022] 5-10 minutes, mobile phase A: 13%vol-16%vol, mobile phase B: 87%vol-84%vol;

[0023] 10-15 minutes, mobile phase A: 16%vol, mobile phase B: 84%vol;

[0024] 15-20 minutes, mobile phase A: 16%vol-20%vol, mobile phase B: 84%vol-80%vol;

[0025] 20-25 minutes, mobile phase A: 20%vol-30%vol, mobile phase B: 80%vol-70%vol;

[0026] 25-26 minutes, mobile phase A: 30%vol-10%vol, mobile phase B: 70%vol-90%vol;

[0027] 26-30 minutes, mobile phase A: 10%vol, mobile phase B: 90%vol.

[0028] Preferably, when performing the ultra performance liquid chromatography determination, the flow rate of the mobile phase is 0.4 mL per minute.

[0029] Preferably, when performing the ultra performance liquid chromatography determination, the detection wavelength is 330 nm.

[0030] Preferably, when performing the ultra performance liquid chromatography determination, the injection volume is 2 μL.

[0031] Preferably, when performing the ultra performance liquid chromatography determination, the column temperature of the chromatographic column is 25℃.

[0032] Preferably, when performing the ultra performance liquid chromatography determination, the theoretical plate number is not less than 3000 calculated according to the peak of verbascoside.

[0033] Preferably, the power of the ultrasonic treatment is 300 W, and the frequency is 40 kHz.

[0034] The application also provides the application of the fingerprint spectrum constructed by the construction method.

[0035] The UPLC fingerprint spectrum corresponding to the test product is subjected to data import, multi-point correction and data matching by using the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, so as to obtain the UPLC control fingerprint spectrum of the test product composed of 9 characteristic peaks;

[0036] The test product is Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) R.Wight medicinal material, decoction piece, standard decoction or formula granule.

[0037] The sample to be identified is prepared into a sample solution, and the sample and the test product are in the same form.

[0038] The sample solution is determined by using the ultra performance liquid chromatography to obtain the UPLC fingerprint spectrum corresponding to the sample;

[0039] The similarity of the UPLC control fingerprint spectrum and the UPLC fingerprint spectrum corresponding to the sample is evaluated by using the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system, and the similarity evaluation result is used as the identification basis of the sample;

[0040] The chromatographic conditions of the ultra performance liquid chromatography and the chromatographic conditions for obtaining the UPLC fingerprint spectrum corresponding to the sample are consistent.

[0041] The application also provides the application of the fingerprint spectrum constructed by the construction method:

[0042] (1) The sample to be identified is used as the sample to obtain the UPLC fingerprint spectrum corresponding to the sample by using the construction method;

[0043] The sample is the Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) O.Kuntze medicinal material, decoction piece, standard decoction or formula granule to be identified.

[0044] (2) The mixture of echinacoside, verbascoside and cistanoside A is used as the control sample to prepare the control sample reference solution;

[0045] The control sample reference solution is determined by using the ultra performance liquid chromatography to obtain the chromatogram of the control sample reference;

[0046] The chromatographic conditions of the ultra performance liquid chromatography and the chromatographic conditions for obtaining the UPLC fingerprint spectrum corresponding to the sample are consistent.

[0047] (3) In the UPLC fingerprint spectrum corresponding to the sample, the peak 5 corresponding to the verbascoside reference peak is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the calculation result is compared with the form of the sample and the range of the specified value, and the corresponding base of the specified value is the base of the sample;

[0048] The range of the specified value of the Cistanche deserticola Y.C.Ma medicinal material is that the relative retention time is within the range of ±10% of the specified value, the specified value is that peak 1 is 0.13, peak 2 is 0.39, peak 6 is 1.06, peak 7 is 1.18, peak 8 is 1.64, peak 9 is 1.79, and the relative peak area is within the specified range, and the specified range is that peak 8 is 0.29-1.30, and peak 9 is 0.17-1.37;

[0049] The range of the prescribed value of the Cistanche tubulosa medicinal material is that the relative retention time is within the range of ±10% of the prescribed value, the prescribed value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; the relative peak area is within the prescribed range, the prescribed range is: peak 8: 0.02-0.06; peak 9: 0.02-0.11;

[0050] The range of the prescribed value of the Cistanche tubulosa medicinal material is that the relative retention time is within the range of ±10% of the prescribed value, the prescribed value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; the relative peak area is within the prescribed range, the prescribed range is: peak 8: 0.02-0.06; peak 9: 0.02-0.11;

[0051] The range of the prescribed value of the Cistanche tubulosa medicinal material is that the relative retention time is within the range of ±10% of the prescribed value, the prescribed value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; the relative peak area is within the prescribed range, the prescribed range is: peak 8: 0.02-0.06; peak 9: 0.02-0.11;

[0052] The range of the prescribed value of the Cistanche tubulosa medicinal material is that the relative retention time is within the range of ±10% of the prescribed value, the prescribed value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; the relative peak area is within the prescribed range, the prescribed range is: peak 8: 0.02-0.06; peak 9: 0.02-0.11;

[0053] The range of the prescribed value of the Cistanche tubulosa medicinal material is that the relative retention time is within the range of ±10% of the prescribed value, the prescribed value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; the relative peak area is within the prescribed range, the prescribed range is: peak 8: 0.02-0.06; peak 9: 0.02-0.11.

[0054] Preferably, the method for preparing the control reference solution is that the control is uniformly mixed with methanol-water. More preferably, 50%vol methanol-water. Preferably, the concentrations of echinacoside and verbascoside in the control reference solution are each 0.2mg / mL, and the concentration of cistanoside A is 0.1mg / L.

[0055] The beneficial effects of the present application are that:

[0056] The application provides a method for establishing UPLC fingerprint spectra of Cistanche or Cistanche tubulosa medicinal materials, decoction pieces, standard decoctions and formula granules.

[0057] The application provides a method for establishing UPLC fingerprint spectra of Cistanche or Cistanche tubulosa medicinal materials, decoction pieces, standard decoctions and formula granules. The method uses a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to establish a control fingerprint spectrum, calibrates nine common characteristic peaks, confirms that peak 1 is cistanchein F, peak 3 is veronicin, peak 4 is cistanchein A, peak 5 is verbascoside, peak 6 is tubuloside A, peak 7 is isorosin, peak 8 is 2'-acetylverbascoside and peak 9 is tubuloside B, selects peak 5, i.e. verbascoside, as an internal reference peak, and determines the relative retention time of the common characteristic peaks of Cistanche or Cistanche tubulosa.

[0058] The method can comprehensively and rapidly detect the quality of Cistanche or Cistanche tubulosa medicinal materials and preparations, is beneficial to comprehensive quality detection and overall quality control of Cistanche or Cistanche tubulosa medicines and preparations, and thus helps to improve the safety, effectiveness and stability of the medicines.

[0059] The application provides a comprehensive and scientific quality standard detection method for Cistanche or Cistanche tubulosa medicines and preparations, including determination of related ingredient contents and fingerprint spectrum detection. The determination method uses accurate parameter control, can completely reflect the quality of Cistanche or Cistanche tubulosa medicines and preparations, and can be used as an evaluation basis for consistency of curative effects of large-scale produced Cistanche or Cistanche tubulosa medicines and preparations, and provides protection for safe and effective and standardized production of Cistanche or Cistanche tubulosa medicines and preparations. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 The figure is a UPLC control fingerprint spectrum of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials. Peak 1 corresponds to cistanchein F; peak 3 corresponds to veronicin; peak 4 corresponds to cistanchein A; peak 5 (S peak) corresponds to verbascoside; peak 6 corresponds to tubuloside A; peak 7 corresponds to isorosin; peak 8 corresponds to 2-acetylverbascoside; and peak 9 corresponds to tubuloside B. 1-9 are serial numbers of the peaks.

[0061] Figure 2 The figure is a UPLC chromatogram (labeled with 20 peaks) of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials obtained in Example 1.

[0062] Figure 3 is the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1 (9 peaks are marked).

[0063] Figure 4 is the Cistancheoside F reference substance positioning (peak 1) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0064] Figure 5 is the Echinacoside reference substance positioning (peak 3) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0065] Figure 6 is the Cistancheoside A reference substance positioning (peak 4) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0066] Figure 7 is the Verbascoside reference substance positioning (peak 5) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0067] Figure 8 is the Tubiflorin A reference substance positioning (peak 6) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0068] Figure 9 is the Isoorientin reference substance positioning (peak 7) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0069] Figure 10 is the 2'-acetylverbascoside reference substance positioning (peak 8) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0070] Figure 11 is the Tubiflorin B reference substance positioning (peak 9) diagram of the UPLC chromatogram of Cistanche (Cistanche deserticola) medicinal material obtained in Example 1.

[0071] Figure 12 is the UPLC control fingerprint chromatogram of Cistanche (Cistanche tubulosa) medicinal material. The peak numbers and corresponding components are the same as Figure 1 .

[0072] Figure 13is the UPLC chromatogram comparison chart of 15 batches of Cistanche (Cistanche tubulosa) medicinal materials in Example 2. The batch numbers of S1-S15 are: 2308001, 2308002, 2308003, 230804, 230805, 230806, 2308007, 2308008, 2308009, 2308010, 2308011, 2308012, 2308013, 2308014, 2308015.

[0073] Figure 14 is the comparison chart of UPLC control fingerprint of Cistanche (Cistanche tubulosa) medicinal materials and UPLC control fingerprint of Cistanche (Cistanche deserticola) medicinal materials in Example 2. In the figure, S1 is the control chart of Cistanche (Cistanche tubulosa) medicinal materials, and S2 is the control chart of Cistanche (Cistanche deserticola) medicinal materials.

[0074] Figure 15 is the UPLC control fingerprint of Cistanche (Cistanche deserticola) decoction pieces. The peak numbers and corresponding components are the same as Figure 1 .

[0075] Figure 16 is the UPLC control fingerprint of Cistanche (Cistanche tubulosa) decoction pieces. The peak numbers and corresponding components are the same as Figure 1 .

[0076] Figure 17 is the UPLC control fingerprint of Cistanche (Cistanche deserticola) standard decoction. The peak numbers and corresponding components are the same as Figure 1 .

[0077] Figure 18 is the UPLC control fingerprint of Cistanche (Cistanche tubulosa) standard decoction. The peak numbers and corresponding components are the same as Figure 1 .

[0078] Figure 19 is the instrument precision chromatogram in Example 9.

[0079] Figure 20 is the repeatability chromatogram in Example 9.

[0080] Figure 21 is the intermediate precision (personnel) chromatogram in Example 9.

[0081] Figure 22 is the negative control chromatogram in Example 9 (4).

[0082] Figure 23 is the stability chromatogram in Example 9.

[0083] Figure 24The chromatogram is obtained under the condition of a flow rate of 0.3 ml / min in Example 9 (6.1).

[0084] Figure 25 The conditions described in Example 9 are: (6.1) a flow rate of 0.4 ml / min; (6.2) a column temperature of 25°C; (6.3) an acetic acid concentration of 0.1%; and (6.4) an Agilent ZORBAX Eclipse Plus C column. 18 The chromatogram obtained under the given conditions.

[0085] Figure 26 The chromatogram is obtained under the condition of a flow rate of 0.5 ml / min in Example 9 (6.1).

[0086] Figure 27 The chromatogram is obtained at a column temperature of 20°C in Example 9 (6.2).

[0087] Figure 28 The chromatogram is obtained at a column temperature of 30°C in Example 9 (6.2).

[0088] Figure 29 The chromatogram is obtained under the condition of acetic acid concentration of 0.08% in Example 9 (6.3).

[0089] Figure 30 The chromatogram is obtained under the condition of acetic acid concentration of 0.12% in Example 9 (6.3).

[0090] Figure 31 The chromatographic column ACQUITY HSS T3 C in Example 9 (6.4) 18 The chromatogram obtained under the given conditions.

[0091] Figure 32 The chromatographic column Shim-pack VeioxC in Example 9 (6.4) 18 The chromatogram obtained under the given conditions. Detailed Implementation

[0092] Experimental instruments and materials:

[0093] Instruments: Thermo Vanquish ultra-high performance liquid chromatograph, including Quaternary Pump F, Split Sampler FT autosampler, Column Compartment H column oven, Diode Array Detector FG detector, and Chromeleon 7 chromatography workstation software;

[0094] Electronic analytical balance: METTLER TOLEDO (Switzerland Mettler) XPE26, MS204S;

[0095] Ultrasonic cleaner: KQ-500DE type Kunshan ultrasonic instrument Co., Ltd.;

[0096] Chromatographic column: Agilent ZORBAX Eclipse Plus C 18 Rapid Resolution HD; 1.8μm 2.1mm×1000mm SN:USDA219376;

[0097] Reagents: acetonitrile for chromatography, water for ultrapure water; other reagents are analytical pure.

[0098] Reagents:

[0099] Echinacoside reference substance (batch number: 111670-201907, for content determination, calculated at 91.8%, purchased from China Institute for Food and Drug Control).

[0100] Verbascoside reference substance (batch number: 111530-201914, for content determination, calculated at 95.2%, purchased from China Institute for Food and Drug Control).

[0101] Cistanoside A (batch number: 93236-42-1, for content determination, calculated at 93.2%, purchased from Chengdu Glipli Biological Technology Co., Ltd.).

[0102] Cistanche (Cistanche deserticola) reference material (batch number: 121101-201603, for thin layer chromatography identification, purchased from China Institute for Food and Drug Control).

[0103] Cistanche (Cistanche tubulosa) reference material (batch number: 121276-201903, for identification / inspection, purchased from China Institute for Food and Drug Control).

[0104] Information of 15 batches of Cistanche (Cistanche deserticola) medicinal materials and 15 batches of Cistanche (Cistanche tubulosa) medicinal materials:

[0105]

[0106] Cistanche, Cistanche tubulosa, standard decoction, formula granules are prepared by Huarun Sanjiu Modern Chinese Medicine Pharmaceutical Co., Ltd. The batch numbers of Cistanche (Cistanche deserticola) are 2111001Y, 2111002Y, 2111003Y, 2111004Y, 2111005Y, 2111006Y, 2111007Y, 2111008Y, 2111009Y, 2111010Y, 2111011Y, 2111012Y, 2111013Y, 2111014Y, 2111015Y. The batch numbers of Cistanche (Cistanche tubulosa) are 2401001Y, 2401002Y, 2401003Y, 2401004Y, 2401005Y, 2401006Y, 2401007Y, 2401008Y, 2401009Y, 2401010Y, 2401011Y, 2401012Y, 2401013Y, 2401014Y, 2401015Y.

[0107] Example 1

[0108] Construction and application of UPLC fingerprint of Cistanche medicinal materials:

[0109] (1) Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% acetic acid solution as mobile phase B, gradient elution according to the provisions in Table 1; column temperature 25°C, flow rate 0.4 ml / min, detection wavelength 330 nm. The theoretical plate number should not be less than 3000 calculated by the peak of verbascoside.

[0110] Table 1 Gradient elution program

[0111]

[0112] Table 2 System suitability parameters

[0113]

[0114] (2) Preparation of test solution: take Cistanche (Cistanche deserticola) medicinal material powder (pass through No. 4 sieve) 1 g, put it in a stoppered conical flask, add water 50 ml, heat reflux for 45 minutes, take out, cool, filter, evaporate the filtrate to dryness, add 50 vol% methanol-water 50 ml, tightly stop, ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes, take out, cool, shake evenly, filter, take the filtrate, and you get it.

[0115] Preparation of reference solution: Take Cistanche (Cistanche deserticola) control medicinal material 1 g, put it in a conical flask with a plug, add water 50 ml, heat reflux for 45 minutes, take it out, cool, filter, evaporate the filtrate to dryness, add 50 vol% methanol-water 50 ml, tightly plug, ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes, take it out, cool, shake evenly, filter, take the filtrate, as the control medicinal material reference solution.

[0116] Preparation of reference solution: Take Cistanche (Cistanche deserticola) control medicinal material 1 g, put it in a conical flask with a plug, add water 50 ml, heat reflux for 45 minutes, take it out, cool, filter, evaporate the filtrate to dryness, add 50 vol% methanol-water 50 ml, tightly plug, ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes, take it out, cool, shake evenly, filter, take the filtrate, as the control medicinal material reference solution.

[0117] (3) Determination method: precisely take 2 μL of the reference solution or the test solution, inject into the liquid chromatograph, determine, and obtain. The flow rate of the mobile phase is 0.4 mL per minute, the detection wavelength is 330 nm, the column temperature of the chromatographic column is 25 ℃, and the theoretical plate number is not less than 3000 according to the peak of verbascoside.

[0118] (4) Establishment of fingerprint and its parameters:

[0119] The UPLC fingerprint of Cistanche (Cistanche deserticola) medicinal material is generated by data import, multi-point correction and data matching of the obtained UPLC spectrum by using the similarity evaluation software of chromatographic fingerprint of traditional Chinese medicines (2012 edition) compiled by the Pharmacopoeia Commission, with multiple batches of representative Cistanche (Cistanche deserticola) medicinal material samples as test samples, as shown in Figure 1 The chemical properties of each characteristic peak of Cistanche (Cistanche deserticola) medicinal material are analyzed, and Cistancheuside F, Echinacoside, Cistancheuside A, Verbascoside, Tubiflorin A, Isoferulic Acid, 2'-Acetyl Verbascoside, and Tubiflorin B are selected for positioning identification.

[0120] (4.1) Confirmation of fingerprint characteristic peaks and selection of S peaks:

[0121] The detection results of the fingerprint of 15 batches of Cistanche (Cistanche deserticola) medicinal material test samples are analyzed, and 20 chromatographic peaks with good separation degree of the obtained UPLC fingerprint of Cistanche (Cistanche deserticola) medicinal material are confirmed by identification of characteristic peaks by UPLC, as shown in Figure 2Some batches of peaks 2, 3, 4, 6, 7, 8, 10, 11, 16, 17, 19 disappeared or were smaller, and the repeatability was poor. Considering the applicability of the fingerprint method in different batches of chromatographic columns and different laboratories, it was not used as a characteristic peak. Peaks 1, 5, 9, 12, 13, 14, 15, 18, 20 were selected as characteristic peaks, and the chromatographic peak numbers were rearranged according to the order of the chromatographic peaks.

[0122] According to the peak identification and the results of the control substance positioning, peak 1 was identified as Cistanche F, peak 3 as Echinacoside, peak 4 as Cistanche A, peak 5 as Verbascoside, peak 6 as Tuberosin A, peak 7 as Isoveronoside, peak 8 as 2'-acetyl Verbascoside, and peak 9 as Tuberosin B. The above identified components belong to phenylethanoid glycosides, and the fingerprint contains the main chemical components of Cistanche (Cistanche deserticola Y.C.Ma), which is consistent with the literature report.

[0123] In the UPLC control fingerprint of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials, Verbascoside had a moderate response and the peak time was in the middle of the whole spectrum, and it was also the content index component of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials. Verbascoside was used as the S peak of the control fingerprint. The relative retention time and relative peak area of each characteristic peak were calculated based on Verbascoside as the S peak.

[0124] (4.2) Confirmation of characteristic peaks of control fingerprint:

[0125] Nine common peaks were selected as characteristic peaks, and through control substance positioning, peak 1 was determined as Cistanche F, peak 3 as Echinacoside, peak 4 as Cistanche A, peak 5 as Verbascoside, peak 6 as Tuberosin A, peak 7 as Isoveronoside, peak 8 as 2'-acetyl Verbascoside, and peak 9 as Tuberosin B. The results are shown in Figures 3-11 .

[0126] (5) Comparison of fingerprints of multiple batches of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials:

[0127] After 15 batches of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials were prepared into test sample solutions, they were determined by ultra-high performance liquid chromatography. The determination results are shown in Tables 3 and 4. The determination results showed that the relative retention times of the characteristic peaks of the 15 batches of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials were within ±10% of the specified value, and should correspond to the retention times of the corresponding 9 characteristic peaks of the Cistanche (Cistanche deserticola Y.C.Ma) control medicinal materials. The relative peak area was within the specified range.

[0128] Table 3 Relative retention time of 15 batches of Cistanche (Cistanche deserticola Y.C.Ma) medicinal materials fingerprint

[0129]

[0130]

[0131] Table 4 Relative peak area of 15 batches of Cistanche (Cistanche deserticola) medicinal materials fingerprint

[0132]

[0133] (6) Verification of the control fingerprint:

[0134] The reference solution of the control sample (corresponding to Cistanche (Cistanche deserticola) control medicinal materials) was determined by ultra-high performance liquid chromatography to obtain the chromatogram of the reference sample;

[0135] The similarity of the chromatogram of the control medicinal material reference and the UPLC control fingerprint of Cistanche (Cistanche deserticola) medicinal materials was evaluated by traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity result was 0.988. The results showed that the control fingerprint generated by the fingerprint of 15 batches of Cistanche (Cistanche deserticola) medicinal materials was consistent with the fingerprint of Cistanche (Cistanche deserticola) control medicinal materials.

[0136] Example 2

[0137] Construction and application of UPLC fingerprint of Cistanche tubulosa medicinal materials:

[0138] The same method as in (1) to (3) in Example 1 was used, except that Cistanche (Cistanche deserticola) medicinal materials were replaced by Cistanche (Cistanche tubulosa) medicinal materials.

[0139] (4) Establishment of fingerprint and its parameters:

[0140] The fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used. Multiple batches of representative Cistanche (Cistanche tubulosa) medicinal material samples were used as test samples. The obtained UPLC chromatogram was data imported, multi-point corrected and data matched to generate the UPLC control fingerprint of Cistanche (Cistanche tubulosa) medicinal materials, as shown in Figure 12 .

[0141] The UPLC fingerprints of each batch of Cistanche (Cistanche tubulosa) medicinal materials were compared, as shown in Figure 13 , and the UPLC control fingerprint of Cistanche (Cistanche tubulosa) medicinal materials and the UPLC control fingerprint of Cistanche (Cistanche deserticola) medicinal materials were compared, as shown in Figure 14 .

[0142] From Figure 14It can be seen that the UPLC standard fingerprint of Cistanche (Cistanche deserticola Y.C.Ma) has 9 common characteristic peaks, which also appear in the UPLC standard fingerprint of Cistanche (Cistanche tubulosa (Schenk) R.Wight), but there is a big difference in the peak area of characteristic peaks 8 and 9 between the two, for example, the response value of the chromatographic peak of peak 8 and peak 9 in the UPLC standard fingerprint of Cistanche (Cistanche tubulosa (Schenk) R.Wight) is significantly lower than that of Cistanche (Cistanche deserticola Y.C.Ma).

[0143] The ratio of the relative peak area of the fingerprint of each batch of Cistanche (Cistanche deserticola Y.C.Ma) and each batch of Cistanche (Cistanche tubulosa (Schenk) R.Wight) is listed as follows:

[0144] Table 5 Relative peak area of 15 batches of Cistanche (Cistanche deserticola Y.C.Ma) fingerprint

[0145]

[0146]

[0147] Table 6 Relative peak area of 15 batches of Cistanche (Cistanche tubulosa (Schenk) R.Wight) fingerprint

[0148]

[0149] From the above results, it can be seen that there is a significant difference in the relative peak area of peaks 8 (Cistanche range: 0.29-1.30, Cistanche tubulosa: 0.02-0.06) and 9 (Cistanche range: 0.17-1.37, Cistanche tubulosa: 0.02-0.11) between the fingerprints of Cistanche (Cistanche deserticola Y.C.Ma) and Cistanche (Cistanche tubulosa (Schenk) R.Wight).

[0150] Therefore, when identifying the origin of medicinal materials, the above relative peak area of the sample to be identified can be compared.

[0151] If the relative retention time is within ±10% of the specified value, the specified value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.06; peak 7: 1.18; peak 8: 1.64, peak 9: 1.79; and the relative peak area is within the specified range, the specified range is: peak 8: 0.29-1.30; peak 9: 0.17-1.37; then it is Cistanche (Cistanche deserticola Y.C.Ma) medicinal material.

[0152] If the relative retention time is within ±10% of the specified value, the specified value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; and the relative peak area is within the specified range, the specified range is: peak 8: 0.02-0.06; peak 9: 0.02-0.11; then it is Cistanche (Cistanche tubulosa (Schenk) R.Wight) medicinal material.

[0153] (6) Verification of the control fingerprint:

[0154] The control reference solution (corresponding to the Cistanche tubulosa control medicinal material) was determined by UPLC to obtain the chromatogram of the control reference solution;

[0155] The similarity of the chromatogram of the control reference solution and the UPLC control fingerprint of the Cistanche tubulosa medicinal material was evaluated by the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicines, and the similarity result was 0.996, indicating that the control fingerprint generated from the fingerprints of the 15 batches of Cistanche tubulosa medicinal materials was consistent with the fingerprint of the Cistanche tubulosa control medicinal material.

[0156] (7) From Figure 14 It can be seen that the different origins (Cistanche deserticola and Cistanche tubulosa control medicinal materials) have obvious differences in the relative peak areas of peaks 8 and 9. The similarity of the chromatograms of the two was evaluated by the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicines, and the similarity result was 0.873.

[0157] Example 3

[0158] Construction of the UPLC fingerprint of Cistanche tubulosa decoction pieces:

[0159] The same method as in (1) to (3) in Example 1 was used, except that the Cistanche tubulosa medicinal material was replaced by Cistanche tubulosa decoction pieces.

[0160] (4) Establishment of the fingerprint and its parameters:

[0161] The similarity evaluation software for chromatographic fingerprints of traditional Chinese medicines 2012 edition prepared by the Pharmacopoeia Commission was used, and multiple batches of representative Cistanche tubulosa decoction pieces were used as test samples. The obtained UPLC chromatograms were data imported, multi-point corrected and data matched to generate the UPLC control fingerprint of Cistanche tubulosa decoction pieces, as shown in Figure 15 .

[0162] Example 4

[0163] Construction of the UPLC fingerprint of Cistanche tubulosa decoction pieces:

[0164] The same method as in Example 3 was used, except that the Cistanche tubulosa decoction pieces were replaced by Cistanche tubulosa decoction pieces.

[0165] The obtained UPLC control fingerprint of Cistanche tubulosa decoction pieces is shown in Figure 16 .

[0166] From Figure 16It can be seen that the UPLC control fingerprint of Cistanche deserticola Y.C.Ma (Cistanche tubulosa) has 9 common characteristic peaks, which also appear in the UPLC standard fingerprint of Cistanche tubulosa (S. Singh) R. R. Rao, but there is a big difference in the peak area of the characteristic peaks between the two, for example: the response value of the chromatographic peak of peak 8 and peak 9 of the UPLC standard fingerprint of Cistanche tubulosa (S. Singh) R. R. Rao is significantly lower than that of Cistanche deserticola Y.C.Ma (Cistanche tubulosa).

[0167] The relative retention time and the ratio of the relative peak area of the fingerprint of each batch of Cistanche deserticola Y.C.Ma (Cistanche tubulosa) and each batch of Cistanche tubulosa (S. Singh) R. R. Rao are listed as follows:

[0168] Table 7 Relative retention time of the fingerprint of 15 batches of Cistanche deserticola Y.C.Ma (Cistanche tubulosa)

[0169]

[0170]

[0171] Table 8 Relative peak area of the fingerprint of 15 batches of Cistanche deserticola Y.C.Ma (Cistanche tubulosa)

[0172]

[0173]

[0174] Table 9 Relative retention time of the fingerprint of 15 batches of Cistanche tubulosa (S. Singh) R. R. Rao

[0175]

[0176]

[0177] Table 10 Relative peak area of the fingerprint of 15 batches of Cistanche tubulosa (S. Singh) R. R. Rao

[0178]

[0179] From the above results, it can be seen that there is a significant difference in the relative peak area of peak 8 and peak 9 between the fingerprint of Cistanche deserticola Y.C.Ma (Cistanche tubulosa) and the fingerprint of Cistanche tubulosa (S. Singh) R. R. Rao. Therefore, when identifying the origin of the decoction pieces, the above relative peak area of the sample to be identified can be compared.

[0180] If the relative retention time is within ±10% of the specified value, the specified value is: peak 1: 0.14; peak 2: 0.40; peak 6: 1.07; peak 7: 1.19; peak 8: 1.60, peak 9: 1.74; and the relative peak area is within the specified range, the specified range is: peak 8: 0.29-1.30; peak 9: 0.17-1.37; then it is Cistanche deserticola Y.C.Ma (Cistanche tubulosa) decoction pieces.

[0181] If the relative retention time is within ±10% of the specified value, the specified value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; and the relative peak area is within the specified range, the specified range is: peak 8: 0.01-0.04; peak 9: 0.02-0.12; then it is Cistanche (Cistanche tubulosa) decoction pieces.

[0182] Example 5

[0183] Construction of Cistanche standard decoction UPLC fingerprint:

[0184] (1) Chromatographic conditions and system suitability test:

[0185] Octadecylsilane-bonded silica gel is used as the filler (column length is 100 mm, inner diameter is 2.1 mm, and particle size is 1.8 μm); acetonitrile is used as mobile phase A, and 0.1% acetic acid solution is used as mobile phase B, gradient elution is carried out according to the provisions in Table 1; the column temperature is 25°C, the flow rate is 0.4 ml per minute, and the detection wavelength is 330 nm. The theoretical plate number calculated according to the peak of verbascoside should not be less than 3000.

[0186] Table 11 System suitability parameters

[0187]

[0188] (2) Preparation of test solution:

[0189] About 0.5 g of Cistanche (Cistanche deserticola) standard decoction powder is accurately weighed, placed in a conical flask with a plug, 50 ml of 50% methanol is accurately added, tightly sealed, shaken uniformly, the weight is determined, ultrasonic treatment (power 300 W, frequency 40 kHz) is carried out for 30 minutes, cooled, the weight is determined again, 50% methanol is added to make up for the weight loss, shaken uniformly, placed, the supernatant is taken, filtered, and the filtrate is taken, which is obtained.

[0190] Preparation of reference solution: an appropriate amount of Echinacoside, Verbascoside, and Cistanchein A reference substances are accurately weighed, 50% methanol solution is added to prepare a mixed solution containing 0.2 mg of Echinacoside, 0.2 mg of Verbascoside, and 0.1 mg of Cistanchein A per 1 ml, which is obtained.

[0191] (3) Determination method: 2 μL of the reference solution or test solution is accurately taken and injected into the liquid chromatograph for determination, which is obtained. The flow rate of the mobile phase is 0.4 mL per minute, the detection wavelength is 330 nm, the column temperature of the chromatographic column is 25°C, and the theoretical plate number calculated according to the peak of Verbascoside is not less than 3000.

[0192] (4) Establishment of fingerprint and its parameters:

[0193] The similarity evaluation software of fingerprint spectrum of traditional Chinese medicine compiled by Pharmacopoeia Commission was used to evaluate the similarity of the fingerprint spectrum. The standard decoction of Cistanche tubulosa (Cistanche tubulosa) was used as the test sample, and the obtained UPLC spectrum was imported, corrected and matched to generate the UPLC control fingerprint spectrum of the standard decoction of Cistanche tubulosa (Cistanche tubulosa), as shown in Figure 17 .

[0194] Example 6

[0195] Construction of UPLC fingerprint spectrum of standard decoction of Cistanche tubulosa:

[0196] The same method as in Example 5 was used, except that the standard decoction of Cistanche tubulosa (Cistanche tubulosa) was replaced by the standard decoction of Cistanche tubulosa (Cistanche tubulosa).

[0197] The UPLC control fingerprint spectrum of the standard decoction of Cistanche tubulosa (Cistanche tubulosa) is shown in Figure 18 .

[0198] As can be seen from Figure 18 , the UPLC control fingerprint spectrum of the standard decoction of Cistanche tubulosa (Cistanche tubulosa) has 9 common characteristic peaks, which also appear in the UPLC standard fingerprint spectrum of the standard decoction of Cistanche tubulosa (Cistanche tubulosa), but there is a large difference in the peak area of the characteristic peaks between the two, for example: the response value of the chromatographic peak of peak 8 and peak 9 of the UPLC standard fingerprint spectrum of the standard decoction of Cistanche tubulosa (Cistanche tubulosa) is significantly lower than that of the standard decoction of Cistanche tubulosa (Cistanche tubulosa).

[0199] The ratio of the relative peak area of the fingerprint spectrum of each batch of the standard decoction of Cistanche tubulosa (Cistanche tubulosa) and each batch of the standard decoction of Cistanche tubulosa (Cistanche tubulosa) is shown in the following table:

[0200] Table 12 Relative peak area of fingerprint spectrum of 15 batches of standard decoction of Cistanche tubulosa (Cistanche tubulosa) slices (freeze-dried powder)

[0201]

[0202] Table 13 Relative peak area of fingerprint spectrum of 15 batches of standard decoction of Cistanche tubulosa (Cistanche tubulosa) slices (freeze-dried powder)

[0203]

[0204]

[0205] As can be seen from the above results, there is a significant difference in the relative peak area of peak 8 and peak 9 between the fingerprint spectrum of the standard decoction of Cistanche tubulosa (Cistanche tubulosa) and the standard decoction of Cistanche tubulosa (Cistanche tubulosa).

[0206] Therefore, in identifying the standard decoction pieces of Cistanche, the relative peak area of the above-mentioned sample to be identified can be compared.

[0207] If the relative retention time is within ±10% of the specified value, the specified value is: peak 1: 0.13; peak 2: 0.40; peak 6: 1.06; peak 7: 1.18; peak 8: 1.62, peak 9: 1.75; and the relative peak area is within the specified range, the specified range is: peak 8: 0.28-1.51; peak 9: 0.11-0.73, then it is the standard decoction pieces of Cistanche (Cistanche deserticola Y.C.Ma).

[0208] If the relative retention time is within ±10% of the specified value, the specified value is: peak 1: 0.13; peak 2: 0.39; peak 6: 1.06; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; and the relative peak area is within the specified range, the specified range is: peak 8: 0.01-0.05; peak 9: 0.02-0.09, then it is the standard decoction pieces of Cistanche (Cistanche tubulosa (Schenh) Woods).

[0209] Example 9

[0210] Analysis method verification:

[0211] (1) Instrument precision test:

[0212] Take the same sample solution (batch number 2111001Y), repeat the injection 6 times according to the same method as in Example 3, and determine the relative retention time and relative peak area of 9 common peaks. The results are shown in Tables 14-15, and the instrument precision chromatogram is shown in Figure 19 The RSD of the relative retention time and relative peak area of each characteristic peak and the reference S peak (peak 5) is small, indicating that the instrument precision is good.

[0213] Table 14 Instrument precision relative retention time test results

[0214]

[0215]

[0216] Table 15 Instrument precision relative peak area test results

[0217]

[0218] (2) Method repeatability test:

[0219] Take 6 batches of Cistanche (Cistanche deserticola Y.C.Ma) powder (batch number 2111001Y), and determine the relative retention time and relative peak area of 9 common peaks according to the same method as in Example 3. The results are shown in Tables 16-17, and the repeatability chromatogram is shown inFigure 20 The relative retention time and relative peak area of each characteristic peak to the reference S peak (peak No. 5) are small, indicating that the method has good repeatability.

[0220] Table 16 Method repeatability relative retention time test results (n = 6)

[0221]

[0222] Table 17 Method repeatability relative peak area test results (n = 6)

[0223]

[0224]

[0225] (3) Intermediate precision (different operators):

[0226] The same batch of Cistanche (Cistanche deserticola Y) decoction pieces (batch number 2111001Y) was determined by two inspectors at different times using the same equipment and the same method as in Example 3, and the results are shown in Tables 18-19, and the intermediate precision (personnel) chromatogram is shown in Figure 21 The relative retention time and relative peak area of each characteristic peak to the reference S peak (peak No. 5) are small, indicating that the method has good intermediate precision.

[0227] Table 18 Intermediate precision relative retention time test results (different operators)

[0228]

[0229] Table 19 Intermediate precision relative peak area test results (different operators)

[0230]

[0231] (4) Specificity:

[0232] The test sample used 50% methanol as the extraction solvent, and 2 μl of the test sample solution and the negative control solution (50% methanol) were precisely taken and injected into the high performance liquid chromatograph, and tested according to the method of Example 3, and the results showed that the negative control had no interference, as shown in Figure 22

[0233] (5) Stability investigation:

[0234] The same batch of test sample (batch number 2111001Y) was prepared, and the relative retention time and relative peak area of 9 common peaks were determined at 0, 4, 8, 12, 18, 24 hours after preparation according to the same method as in Example 3, and the results are shown in Tables 20-21, and the stability chromatogram is shown in Figure 23 ​The relative retention time and relative peak area of each characteristic peak to the reference substance S peak were small, indicating that each peak of the test solution was stable within 24 hours, meeting the determination requirements.

[0235] Table 20 Stability relative retention time test results

[0236]

[0237] Table 21 Stability relative peak area test results

[0238]

[0239] (6) Durability:

[0240] (6.1) Investigation of different flow rates:

[0241] The same batch of test solution (batch number 2111001Y) was used, and the determination method was the same as in Example 3. The flow rate was set to 0.3 ml / min, 0.4 ml / min, and 0.5 ml / min to determine the separation effect of each characteristic peak when the flow rate changed. The results are shown in Tables 22-23, Figures 24-26 The results showed that the separation effect of each characteristic peak was good at different flow rates. However, the relative retention time deviation was large at different flow rates.

[0242] Table 22 Relative retention time results at different flow rates

[0243]

[0244]

[0245] Table 23 Relative peak area results at different flow rates

[0246]

[0247] (6.2) Investigation of different column temperatures:

[0248] The same batch of test solution (batch number 2111001Y) was used, and the determination method was the same as in Example 3. The column temperature was set to 20℃, 25℃, and 30℃ to investigate the separation effect of each characteristic peak when the column temperature changed slightly. The results are shown in Tables 24-25, Figure 27 25

[0249] Table 24 Comparison of determination results at different column temperatures

[0250] ​​

[0251] Table 25 Relative peak area results of different column temperatures

[0252]

[0253] (6.3) Investigation of different mobile phases:

[0254] The same batch of test solution (batch number 2111001Y) was used, and the same determination method as in Example 3 was used to set the acetic acid concentration of the mobile phase to 0.08%, 0.1%, and 0.12%, respectively, to investigate the separation effect of each characteristic peak when acetic acid changes slightly, and the results are shown in Tables 26-27, Figure 29 25 , 30. The results show that when the acetic acid concentration changes slightly, the separation degree of each characteristic peak is good. This method has good robustness for different acetic acid concentrations.

[0255] Table 26 Comparison of determination results at different acetic acid concentrations

[0256]

[0257]

[0258] Table 27 Relative peak area results at different acetic acid concentrations

[0259]

[0260] (6.4) Investigation of different chromatographic columns:

[0261] The same batch of test solution (batch number 2111001Y) was used, and the same determination method as in Example 3 was used to set different types of chromatographic columns to ACQUITY HSS T3-C 18 , Agilent ZORBAX Eclipse Plus C 18 Rapid Resolution HD, and Shim-pack Veiox-C 18 , to investigate the separation effect and retention of each characteristic peak when the chromatographic column changes, and the results are shown in Tables 28-29, Figure 31 25 , 32. The results show that the retention time and peak area of each characteristic chromatographic peak are affected by the type of chromatographic column, especially the peak shape of chromatographic peak 1 on the Shim-pack Veiox-C 18 chromatographic column is poor, so it is recommended to use the Agilent ZORBAX Eclipse Plus C 18 Rapid Resolution HD (2.1 mm x 100 mm, 1.8 μm) as the chromatographic column for research and investigation. ​​

[0262] Table 28 Comparison of results from different chromatographic columns

[0263]

[0264] Table 29 Relative peak area results from different chromatographic columns

[0265]

Claims

1. A method for constructing UPLC fingerprint spectra of Cistanche deserticola or Cistanche tubulosa medicinal materials, processed slices, standard decoctions, and formulated granules, characterized in that, Includes the following steps: Preparation of test samples: The test samples are Cistanche deserticola or Cistanche tubulosa medicinal materials, decoction pieces, standard decoctions or formula granules; The test sample is prepared into a test sample solution; When the test sample is Cistanche deserticola or Cistanche tubulosa medicinal material or processed slices, the method for preparing the test sample into a test sample solution is as follows: The test sample was placed in water and heated under reflux for 45 minutes. After cooling, it was filtered and the filtrate was evaporated to dryness. Then, methanol-water was added, and the mixture was sonicated for 30-60 minutes. After cooling, it was filtered again, and the filtrate was collected. When the test sample is a standard decoction or granule of Cistanche deserticola or Cistanche tubulosa, the method for preparing the test sample into a test sample solution is as follows: Place the test sample in methanol-water and sonicate for 15-60 minutes. After cooling, add methanol-water to make up the weight loss. Mix well and let stand. Take the supernatant and filter it. Take the filtrate to obtain the test sample. The test solution was analyzed by ultra-high performance liquid chromatography to obtain the UPLC fingerprint spectrum corresponding to the test sample. The chromatographic conditions for the ultra-high performance liquid chromatography (UHPLC) were as follows: Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD column; detection wavelength: 330 nm; mobile phase A: acetonitrile; mobile phase B: 0.1 wt% acetic acid solution; gradient elution. The gradient elution procedure is as follows: 0–5 minutes, mobile phase A: 10% vol–13% vol, mobile phase B: 90% vol–87% vol; 5–10 minutes, mobile phase A: 13% vol–16% vol, mobile phase B: 87% vol–84% vol; 10–15 minutes, mobile phase A: 16% vol, mobile phase B: 84% vol; 15–20 minutes, mobile phase A: 16% vol–20% vol, mobile phase B: 84% vol–80% vol; 20–25 minutes, mobile phase A: 20% vol–30% vol, mobile phase B: 80% vol–70% vol; 25–26 minutes, mobile phase A: 30% vol–10% vol, mobile phase B: 70% vol–90% vol; 26–30 minutes, mobile phase A: 10% vol, mobile phase B: 90% vol; The reference standards include: echinacoside, verbascoside, cistancheside A, cistancheside F, tubuloside A, iso-citricascoside, 2'-acetylverascoside, and tubuloside B.

2. The construction method according to claim 1, characterized in that, The prepared slices of Cistanche deserticola or Cistanche tubulosa are processed products of Cistanche deserticola or Cistanche tubulosa medicinal materials; the standard decoction of Cistanche deserticola or Cistanche tubulosa is a freeze-dried powder prepared from Cistanche deserticola or Cistanche tubulosa medicinal materials through processing, decoction, separation, concentration, and freeze-drying; the granules of Cistanche deserticola or Cistanche tubulosa are granules made from Cistanche deserticola or Cistanche tubulosa medicinal materials through processing, extraction, separation, concentration, drying, granulation, and packaging processes.

3. The construction method according to claim 1, characterized in that, The solvent for the test solution is methanol-water.

4. The construction method according to claim 1, characterized in that, The solvent for the test solution is 50% vol methanol-water.

5. The construction method according to claim 1, characterized in that, In the test solution, the ratio of test sample to solvent is 1g:50mL.

6. The construction method according to claim 1, characterized in that, When performing the ultra-high performance liquid chromatography (UHPLC) determination, the mobile phase flow rate was 0.4 mL per minute, the injection volume was 2 μL, and the column temperature was 25 °C.

7. The construction method according to claim 1, characterized in that, When performing the ultra-high performance liquid chromatography determination, the theoretical plate number calculated based on the verbascoside peak should be no less than 3000.

8. The construction method according to claim 1, characterized in that, The ultrasonic treatment has a power of 300W and a frequency of 40kHz.

9. The application of the fingerprint spectrum constructed by the construction method according to any one of claims 1-8, characterized in that, include: Using a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, data import, multi-point correction, and data matching were performed on the UPLC fingerprint chromatograms corresponding to the test sample to obtain a UPLC reference fingerprint chromatogram of the test sample consisting of 9 characteristic peaks. The test sample is Cistanche deserticola or Cistanche tubulosa medicinal material, processed slices, standard decoction or formula granules; The sample to be identified is prepared into a sample solution, wherein the sample is in the same form as the test sample; The sample solution was analyzed by ultra-high performance liquid chromatography to obtain the UPLC fingerprint spectrum corresponding to the sample. The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine is used to evaluate the similarity between the UPLC reference fingerprint and the UPLC fingerprint corresponding to the sample, and the similarity evaluation results are used as the basis for the identification of the sample. The chromatographic conditions for the ultra-high performance liquid chromatography (UHPLC) and the chromatographic conditions for obtaining the UPLC fingerprint chromatogram corresponding to the test sample are consistent.

10. The application of the fingerprint spectrum constructed by the construction method according to any one of claims 1-8, characterized in that, include: (1) Using the construction method described above, the sample to be identified is used as the test sample to obtain the UPLC fingerprint spectrum corresponding to the sample; The samples were the medicinal materials, processed slices, standard decoctions, or formula granules of Cistanche deserticola or Cistanche tubulosa to be identified. (2) Prepare a reference solution by using a mixture of echinacoside, verbascoside and cistancheside A as a reference standard; The reference solution was analyzed by ultra-high performance liquid chromatography to obtain a chromatogram of the reference solution. The chromatographic conditions for the ultra-high performance liquid chromatography method and the chromatographic conditions for obtaining the UPLC fingerprint spectrum corresponding to the test sample are consistent. (3) In the UPLC fingerprint spectrum corresponding to the sample, peak 5 corresponding to the peak of verbascoside reference is peak S. Calculate the relative retention time of each characteristic peak and peak S. Compare the calculation results with the form of the sample and the range of the specified values. If the original material corresponds to the specified value, it is the original material of the sample. The specified values ​​for Cistanche deserticola medicinal materials are as follows: relative retention time within ±10% of the specified value, with the specified values ​​being: peak 1: 0.13; peak 2: 0.39; peak 6: 1.06; peak 7: 1.18; peak 8: 1.64, peak 9: 1.79; and relative peak area within the specified range, with the specified range being: peak 8: 0.29~1.30; peak 9: 0.17~1.

37. The specified values ​​for Cistanche tubulosa medicinal materials are as follows: relative retention time within ±10% of the specified value, with the specified values ​​being: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; and relative peak area within the specified range, with the specified range being: peak 8: 0.02~0.06; peak 9: 0.02~0.

11. The specified values ​​for Cistanche deserticola slices are as follows: relative retention time within ±10% of the specified value, with the specified values ​​being: peak 1: 0.14; peak 2: 0.40; peak 6: 1.07; peak 7: 1.19; peak 8: 1.60, peak 9: 1.74; and relative peak area within the specified range, with the specified range being: peak 8: 0.29~1.30; peak 9: 0.17~1.

37. The specified values ​​for the Cistanche tubulosa slices are as follows: relative retention time within ±10% of the specified value, with the specified values ​​being: peak 1: 0.13; peak 2: 0.39; peak 6: 1.05; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; and relative peak area within the specified range, with the specified range being: peak 8: 0.01~0.04; peak 9: 0.02~0.

12. The specified values ​​for the standard decoction or formula granules of Cistanche deserticola are as follows: the relative retention time is within ±10% of the specified value, and the specified values ​​are: peak 1: 0.13; peak 2: 0.40; peak 6: 1.06; peak 7: 1.18; peak 8: 1.62, peak 9: 1.75; the relative peak area is within the specified range, and the specified range is: peak 8: 0.28~1.51; peak 9: 0.11~0.

73. The specified values ​​for the standard decoction or formula granules of Cistanche tubulosa are as follows: the relative retention time is within ±10% of the specified value, and the specified values ​​are: peak 1: 0.13; peak 2: 0.39; peak 6: 1.06; peak 7: 1.17; peak 8: 1.65, peak 9: 1.78; the relative peak area is within the specified range, and the specified range is: peak 8: 0.01~0.05; peak 9: 0.02~0.

09.

11. The application according to claim 10, characterized in that, The method for preparing the reference standard into a reference solution is as follows: the reference standard is uniformly mixed with methanol-water.

12. The application according to claim 10, characterized in that, The methanol-water mixture is 50% vol methanol-water.

13. The application according to claim 10, characterized in that, The concentrations of echinacoside and verbascoside in the reference solution were each 0.2 mg / mL, and the concentration of cistanche glycoside A was 0.1 mg / L.

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